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Purpose: Activation of the mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol 3-kinase/AKT pathway seems to be critical for melanoma proliferation. Components of these pathways are client proteins of heat-shock protein 90 (hsp90), suggesting that inhibition of hsp90 could have significant antimelanoma effects. We conducted a phase II trial using the hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) in melanoma patients. The primary end points were clinical responses and whether treatment inhibited MAPK pathway activity.Experimental Design: Melanoma patients with measurable disease were stratified on the basis of whether or not their tumor harbored a V600E BRAF mutation. The hsp90 inhibitor 17-AAG was administered i.v. once weekly ×6 weeks at 450 mg/m2. Tumor biopsies were obtained pretreatment and 18 to 50 hours after the first dose of 17-AAG, and were snap-frozen.Results: Fifteen evaluable patients were treated; nine had BRAF mutations and six were wild-type. No objective responses were observed. Western blot analysis of tumor biopsies showed an increase in hsp70 and a decrease in cyclin D1 expression in the posttreatment biopsies but no significant effect on RAF kinases or phospho–extracellular signal-regulated kinase expression. Plasma analyzed by mutant-specific PCR for V600E BRAF showed 86% sensitivity and 67% specificity in predicting tumor DNA sequencing results.Conclusions: At this dose and schedule of 17-AAG, the effects of 17-AAG on RAF kinase expression were short-lived, and no objective antimelanoma responses were seen. Future trials in melanoma should focus on a more potent hsp90 inhibitor or a formulation that can be administered chronically for a more prolonged suppression of the MAPK pathway.

Purpose: Both phosphatidylinositol 3-kinase/AKT and RAS/mitogen-activated protein kinase signal transduction pathways mediate 4E-BP1 phosphorylation, releasing 4E-BP1 from the mRNA cap and permitting translation initiation. Given the prevalence of PTEN and BRAF mutations in melanoma, we first examined translation initiation, as measured by phosphorylated 4E-BP1 (p-4E-BP1), in metastatic melanoma tissues and cell lines. We then tested the association between amounts of total and p-4E-BP1 and patient survival.Experimental Design: Seven human metastatic melanoma cells lines and 72 metastatic melanoma patients with accessible metastatic tumor tissues and extended follow-up information were studied. Expression of 4E-BP1 transcript, total 4E-BP1 protein, and p-4E-BP1 was examined. The relationship between 4E-BP1 transcript and protein expression was assessed in a subset of patient tumors (n = 41). The association between total and p-4E-BP1 levels and survival was examined in the larger cohort of patients (n = 72).Results: 4E-BP1 was hyperphosphorylated in 4 of 7 melanoma cell lines harboring both BRAF and PTEN mutations compared with untransformed melanocytes or RAS/RAF/PTEN wild-type melanoma cells. 4E-BP1 transcript correlated with 4E-BP1 total protein levels as measured by the semiquantitative reverse-phase protein array (P = 0.012). High levels of p-4E-BP1 were associated with worse overall and post-recurrence survival (P = 0.02 and 0.0003, respectively).Conclusion: Our data show that translation initiation is a common event in human metastatic melanoma and correlates with worse prognosis. Therefore, effective inhibition of the pathways responsible for 4E-BP1 phosphorylation should be considered to improve the treatment outcome of metastatic melanoma patients.

Purpose: In certain cancers, MDM2 SNP309 has been associated with early tumor onset in women. In melanoma, incidence rates are higher in women than in men among individuals less than 40 years of age, but among those older than 50 years of age, melanoma is more frequent in men than in women. To investigate this difference, we examined the association among MDM2 SNP309, age at diagnosis, and gender among melanoma patients.Experimental Design: Prospectively enrolled melanoma patients (N = 227) were evaluated for MDM2 SNP309 and the related polymorphism, p53 Arg72Pro. DNA was isolated from patient blood samples, and genotypes were analyzed by PCR-restriction fragment length polymorphism. Associations among MDM2 SNP309, p53 Arg72Pro, age at diagnosis, and clinicopathologic features of melanoma were analyzed.Results: The median age at diagnosis was 13 years earlier among women with a SNP309 GG genotype (46 years) compared with women with TG+TT genotypes (59 years; P = 0.19). Analyses using age dichotomized at each decade indicated that women with a GG genotype had significantly higher risks of being diagnosed with melanoma at ages P = 0.01). Similar observations were not seen among men.Conclusions: Our data suggest that MDM2 may play an important role in the development of melanoma in women. The MDM2 SNP309 genotype may help identify women at risk of developing melanoma at a young age.

Supplementary Data from Phase II Trial of 17-Allylamino-17-Demethoxygeldanamycin in Patients with Metastatic Melanoma

Supplementary Table S1 from Association of MDM2 SNP309, Age of Onset, and Gender in Cutaneous Melanoma

Supplementary Table 4 from Integrative Genomics Identifies Molecular Alterations that Challenge the Linear Model of Melanoma Progression

Supplementary Methods from Integrative Genomics Identifies Molecular Alterations that Challenge the Linear Model of Melanoma Progression

Supplementary Table 5 from Integrative Genomics Identifies Molecular Alterations that Challenge the Linear Model of Melanoma Progression

Supplementary Table 2 from Integrative Genomics Identifies Molecular Alterations that Challenge the Linear Model of Melanoma Progression

Supplementary Table 3 from Integrative Genomics Identifies Molecular Alterations that Challenge the Linear Model of Melanoma Progression

Supplementary Table 1 from Integrative Genomics Identifies Molecular Alterations that Challenge the Linear Model of Melanoma Progression

Supplementary Table 6 from Integrative Genomics Identifies Molecular Alterations that Challenge the Linear Model of Melanoma Progression

Introduction: Nevisense is a non-invasive device that measures electrical impedance spectroscopy (EIS) of individual skin lesions to aid in the diagnosis of melanoma. While EIS has demonstrated high sensitivity in diagnosing melanoma, its impact on a clinician’s diagnostic confidence remains unknown. Objective: To conduct a pilot study to evaluate whether clinician diagnostic confidence, sensitivity, specificity and accuracy can be increased by adding EIS measurement scores to clinical and dermoscopic images of lesions clinically suspicious for melanoma. Methods: Three pigmented lesions specialists and three 4th year medical students completed an online survey to evaluate 34 melanocytic lesions suspicious for melanoma. For each lesion, participants provided their diagnosis, biopsy recommendation, and confidence in diagnosing a lesion as benign or malignant based on history and clinical and dermoscopic images, and again after receiving an EIS score. Results: Addition of EIS scores increased mean biopsy sensitivity for melanoma/severely dysplastic nevi from 70% to 84% (p = .014) and mean diagnostic accuracy from 74% to 86% (p = .005). Mean diagnostic confidence increased for all histopathologic categories for both students and dermatologists (all p < .05). Conclusions: In this pilot study, EIS increased novice and expert diagnosticians’ confidence regarding dermoscopically equivocal melanocytic lesions. Further studies are needed to explore how EIS can help clinicians reassure patients regarding the management of clinically dysplastic melanocytic nevi.

Survival outcomes in melanoma and their association with mutations in the telomerase reverse transcriptase gene TERT promoter remain uncertain. In addition, few studies have examined whether these associations are affected by a nearby common germline polymorphism or vary on the basis of melanoma histopathological subtype. We analyzed 408 primary tumors from a prospective melanoma cohort for somatic TERT

Detecting mutations in the plasma of patients with solid tumors is becoming a valuable method of diagnosing and monitoring cancer. The TERT promoter is mutated at high frequencies in multiple cancer types, most commonly at positions -124 and -146 (designated C228T and C250T, respectively). Detection of these mutations has been challenging because of the high GC content of this region (approximately 80%). We describe development of novel probe-based droplet digital PCR assays that specifically detect and quantify these two mutations, along with the less common 242-243 CC>TT mutation, and demonstrate their application using human tumor and plasma samples from melanoma patients. Assay designs and running conditions were optimized using cancer cell line genomic DNAs with the C228T or C250T mutations. The limits of detection were 0.062% and 0.051% mutant allele fraction for the C228T and C250T assays, respectively. Concordance of 100% was observed between droplet digital PCR and sequencing-based orthogonal methods in the detection of TERT mutant DNA in 32 formalin-fixed, paraffin-embedded melanoma tumors. TERT(mutant) DNA was also identified in 21 of 27 plasma samples (78%) from patients with TERT(mutant) tumors, with plasma mutant allele fractions ranging from 0.06% to 15.3%. There were no false positives in plasma. These data demonstrate the potential of these assays to specifically detect and quantify TERT(mutant) DNA in tumors and plasma of cancer patients.

Background: Analysis of cell-free circulating tumor DNA (ctDNA) from blood provides an opportunity to detect potential mechanisms of resistance to targeted therapies in melanoma patients. Approximately 20% and 17% of patients undergoing BRAF targeted therapy develop resistance associated with copy number increases in either BRAF or cylinD1 (CCND1), respectively. Detection of BRAF or CCND1 copy number gains prior to radiographic tumor progression could potentially prompt a therapeutic switch aimed at treating patients with smaller tumors burdens, which may improve survival. Conventional droplet digital PCR (ddPCR) for copy number analysis uses two probes, one target and one reference gene (2-plex). To improve the accuracy and sensitivity of the copy number analysis and account for possible genetic alterations in the reference gene, we performed a 5-probe multiplex (5-plex) reaction using three reference genes and two targets. Methods: We used cell lines with known copy number gains for the BRAF gene (M395-R) and CCND1 gene (SkMel-187) to identify copy number increases using ddPCR. To estimate the limit of detection of the copy number gains in plasma, high and low concentrations of the cell line DNAs were spiked into healthy plasma and divided into three replicate samples of 3ml each. DNA was extracted from plasma using the Circulating DSP NA Kit (Qiagen). The ddPCR reaction mix used 4X Supermix and five different copy number specific probes targeting: two genes of interest, BRAF (7q34) and CCND1 (11q13.3); and three control genes: FOXI3 (2p11.2), AGO1 (1p34.3) and TTC5 (14q11.2). Each probe was titrated to display a unique cluster location on the 2D plot. Probe concentrations were: BRAF: FAM 2X; CCND1: FAM 1X; FOXI3: HEX 2X; AGO1: HEX 1X; TTC5: FAM 0.7X/HEX 0.4X. Each extraction was divided into 4 replicate wells. Following droplet generation, gene copies were analyzed using the QX Manager 1.2 Standard Edition for advanced analysis. Results: Probe titration resulted in 32 quantifiably distinct ddPCR clusters on a single 2D plot. Copies/ul of the reference genes were used as a benchmark for diploid status. We determined that the M395-R cell line had 45 copies of the BRAF gene and that SKMel-187 had eight copies of the CCND1 gene compared to diploid PBMC DNA (Promega). Based on the spike-in experiments, we estimate that we can detect copy number gains in plasmas with 10% or 20% tumor fraction when tumor cells have approximately 45 or 8 copies of the target gene, respectively. Conclusion: We successfully multiplexed five copy number assays in a single ddPCR reaction, and showed that copy number changes could be identifiable with as little as 10% tumor fraction within plasma when the tumor-associated copy number gains were relatively high. Overall, the 5-plex assay has the potential to be excellent tool for copy number detection in blood, and can be customized as needed for other targets. Citation Format: Jennifer M. Wiggins, Mahrukh Syeda, David Polsky. Development of a novel 5-plex copy number ddPCR assay for ctDNA analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 539.

A diagnosis of late-stage melanoma is associated with significant mortality. From a public health perspective, the knowledge of geographic disparities in late-stage diagnoses can inform efforts to facilitate the diagnosis of the earlier stage, highly curable melanomas. We conducted a county-level analysis of melanoma in New York state to identify communities that may benefit from pilot health interventions to reduce the burden of late-stage melanoma. From 1995 to 2016, late-stage melanoma incidence increased from 1.5 to 2.8 cases per 100,000 in New York state. We found statistically significant associations between decreased county-level health system access (including physician density and resident educational status) and increased county incidence and proportion of late-stage disease among diagnosed cases (P0.001 for both). Increased county-level socioeconomic status, including measures of resident wealth and medical insurance status, was positively associated with greater late-stage incidence (P0.001). However, decreased county-level socioeconomic status was positively associated with a greater proportion of late-stage disease among cases at diagnosis (P = 0.009). Counties with reduced access to physician services and lower socioeconomic status may be suitable for pilot interventions promoting the recognition and diagnosis of early-stage melanomas to reduce late-stage diagnoses and associated mortality.

IMPORTANCE: Use of prognostic gene expression profile (GEP) testing in cutaneous melanoma (CM) is rising despite a lack of endorsement as standard of care. OBJECTIVE: To develop guidelines within the national Melanoma Prevention Working Group (MPWG) on integration of GEP testing into the management of patients with CM, including (1) review of published data using GEP tests, (2) definition of acceptable performance criteria, (3) current recommendations for use of GEP testing in clinical practice, and (4) considerations for future studies. EVIDENCE REVIEW: The MPWG members and other international melanoma specialists participated in 2 online surveys and then convened a summit meeting. Published data and meeting abstracts from 2015 to 2019 were reviewed. FINDINGS: The MPWG members are optimistic about the future use of prognostic GEP testing to improve risk stratification and enhance clinical decision-making but acknowledge that current utility is limited by test performance in patients with stage I disease. Published studies of GEP testing have not evaluated results in the context of all relevant clinicopathologic factors or as predictors of regional nodal metastasis to replace sentinel lymph node biopsy (SLNB). The performance of GEP tests has generally been reported for small groups of patients representing particular tumor stages or in aggregate form, such that stage-specific performance cannot be ascertained, and without survival outcomes compared with data from the American Joint Committee on Cancer 8th edition melanoma staging system international database. There are significant challenges to performing clinical trials incorporating GEP testing with SLNB and adjuvant therapy. The MPWG members favor conducting retrospective studies that evaluate multiple GEP testing platforms on fully annotated archived samples before embarking on costly prospective studies and recommend avoiding routine use of GEP testing to direct patient management until prospective studies support their clinical utility. CONCLUSIONS AND RELEVANCE: More evidence is needed to support using GEP testing to inform recommendations regarding SLNB, intensity of follow-up or imaging surveillance, and postoperative adjuvant therapy. The MPWG recommends further research to assess the validity and clinical applicability of existing and emerging GEP tests. Decisions on performing GEP testing and patient management based on these results should only be made in the context of discussion of testing limitations with the patient or within a multidisciplinary group.

Objectives. To determine the effect of new therapies and trends toward reduced mortality rates of melanoma. Methods. We reviewed melanoma incidence and mortality among Whites (the group most affected by melanoma) in 9 US Surveillance, Epidemiology, and End Results registry areas that recorded data between 1986 and 2016. Results. From 1986 to 2013, overall mortality rates increased by 7.5%. Beginning in 2011, the US Food and Drug Administration approved 10 new treatments for metastatic melanoma. From 2013 to 2016, overall mortality decreased by 17.9% (annual percent change [APC] = −6.2%; 95% confidence interval [CI] = −8.7%, −3.7%) with sharp declines among men aged 50 years or older (APC = −8.3%; 95% CI = −12.2%, −4.1%) starting in 2014. This recent, multiyear decline is the largest and most sustained improvement in melanoma mortality ever observed and is unprecedented in cancer medicine. Conclusions. The introduction of new therapies for metastatic melanoma was associated with a significant reduction in population-level mortality. Future research should focus on developing even more effective treatments, identifying biomarkers to select patients most likely to benefit, and renewing emphasis on public health approaches to reduce the number of patients with advanced disease.

BACKGROUND: Little is known on whether melanocortin 1 receptor (MC1R) associated cutaneous melanoma (CM) risk varies depending on histological subtype and body site, and whether tumour thickness at diagnosis (the most important prognostic factor for CM patients) differs between MC1R variant carriers and wild-type individuals. OBJECTIVE: We studied the association between MC1R variants and CM risk by histological subtype, body site, and Breslow thickness, using the database of the M-SKIP project. METHODS: We pooled individual data from fifteen case-control studies conducted during 2005–2015 in Europe and the USA. Study-specific, multi-adjusted odds ratios were pooled into summary odds ratios (SOR) and 95% confidence intervals (CI) using random-effects models. RESULTS: 6891 CM cases and 5555 controls were included. CM risk was increased among MC1R variant carriers vs. wild-type individuals. The increase in risk was comparable across histological subtypes (SOR for any variant vs. wild-type ranged between 1.57 and 1.70, always statistical significant) except acral lentiginous melanoma, for which no association emerged; and slightly greater on chronically (1.74, 95% CI 1.47–2.07) than intermittently (1.55, 95% CI 1.34–1.78) sun-exposed skin. CM risk was greater for those carrying ‘R’ vs. ‘r’ variants; correlated with the number of variants; and was more evident among individuals not showing the red hair colour phenotype. Breslow thickness was not associated with MC1R status. CONCLUSION: MC1R variants were associated with an increased risk of CM of any histological subtype (except ALM) and occurring on both chronically and intermittently sun-exposed skin.

This article, co-authored by a patient diagnosed with acral melanoma, discusses the patient’s experience of being diagnosed with and treated with surgery for this disease. The physician discusses the epidemiology, genetics, diagnosis, treatment, and prognosis of acral melanoma. Follow-up care plans are also discussed.

Melanoma represents an important public health problem, due to its high case-fatality rate. Identification of individuals at high risk would be of major interest to improve early diagnosis and ultimately survival. The aim of this study was to evaluate whether MC1R variants predicted melanoma risk independently of at-risk phenotypic characteristics.Data were collected within an international collaboration - the M-SKIP project. The present pooled analysis included data on 3,830 single, primary, sporadic, cutaneous melanoma cases and 2,619 controls from seven previously published case-control studies. All the studies had information on MC1R gene variants by sequencing analysis and on hair color, skin phototype, and freckles, ie, the phenotypic characteristics used to define the red hair phenotype.The presence of any MC1R variant was associated with melanoma risk independently of phenotypic characteristics (OR 1.60; 95% CI 1.36-1.88). Inclusion of MC1R variants in a risk prediction model increased melanoma predictive accuracy (area under the receiver-operating characteristic curve) by 0.7% over a base clinical model (P=0.002), and 24% of participants were better assessed (net reclassification index 95% CI 20%-30%). Subgroup analysis suggested a possibly stronger role of MC1R in melanoma prediction for participants without the red hair phenotype (net reclassification index: 28%) compared to paler skinned participants (15%).The authors suggest that measuring the MC1R genotype might result in a benefit for melanoma prediction. The results could be a valid starting point to guide the development of scientific protocols assessing melanoma risk prediction tools incorporating the MC1R genotype.

Background The American Joint Committee on Cancer (AJCC) maintains that the eighth edition of its Staging Manual (AJCC8) has improved accuracy compared with the seventh (AJCC7). However, there are concerns that implementation may disrupt analysis of active clinical trials for stage III patients. We used an independent cohort of melanoma patients to test the extent to which AJCC8 has improved prognostic accuracy compared with AJCC7. Methods We analyzed a cohort of 1315 prospectively enrolled patients. We assessed primary tumor and nodal classification of stage I–III patients using AJCC7 and AJCC8 to assign disease stages at diagnosis. We compared recurrence-free (RFS) and overall survival (OS) using Kaplan-Meier curves and log-rank tests. We then compared concordance indices of discriminatory prognostic ability and area under the curve of 5-year survival to predict RFS and OS. All statistical tests were two-sided. Results Stage IIC patients continued to have worse outcomes than stage IIIA patients, with a 5-year RFS of 26.5% (95% confidence interval [CI] = 12.8% to 55.1%) vs 56.0% (95% CI = 37.0% to 84.7%) by AJCC8 (P = .002). For stage I, removing mitotic index as a T classification factor decreased its prognostic value, although not statistically significantly (RFS concordance index [C-index] = 0.63, 95% CI = 0.56 to 0.69; to 0.56, 95% CI = 0.49 to 0.63, P = .07; OS C-index = 0.48, 95% CI = 0.38 to 0.58; to 0.48, 95% CI = 0.41 to 0.56, P = .90). For stage II, prognostication remained constant (RFS C-index = 0.65, 95% CI = 0.57 to 0.72; OS C-index = 0.61, 95% CI = 0.50 to 0.72), and for stage III, AJCC8 yielded statistically significantly enhanced prognostication for RFS (C-index = 0.65, 95% CI = 0.60 to 0.70; to 0.70, 95% CI = 0.66 to 0.75, P = .01). Conclusions Compared with AJCC7, we demonstrate that AJCC8 enables more accurate prognosis for patients with stage III melanoma. Restaging a large cohort of patients can enhance the analysis of active clinical trials.

Mutational heterogeneity can contribute to therapeutic resistance in solid cancers. In melanoma, the frequencies of intertumoral and intratumoral heterogeneity are controversial. We examined mutational heterogeneity within individual patients with melanoma using multiplatform analysis of commonly mutated driver and nonpassenger genes. We analyzed paired primary and metastatic tumors from 60 patients and multiple metastatic tumors from 39 patients whose primary tumors were unavailable (n = 271 tumors). We used a combination of multiplex SNaPshot assays, Sanger sequencing, mutation-specific PCR, or droplet digital PCR to determine the presence of BRAFV600, NRASQ61, TERT–124C>T, and TERT–146C>T mutations. Mutations were detected in BRAF (39%), NRAS (21%), and/or TERT (78%). Thirteen patients had TERTmutant discordant tumors; seven of these had a single tumor with both TERT–124C>T and TERT–146C>T mutations present at different allele frequencies. Two patients had both BRAF and NRAS mutations; one had different tumors and the other had a single tumor with both mutations. One patient with a BRAFmutant primary lacked mutant BRAF in at least one of their metastases. Overall, we identified mutational heterogeneity in 18 of 99 patients (18%). These results suggest that some primary melanomas may be composed of subclones with differing mutational profiles. Such heterogeneity may be relevant to treatment responses and survival outcomes.

BACKGROUND: Patients diagnosed with metastatic melanoma have varied clinical courses, even in patients with similar disease characteristics. We examine the impact of initial stage of melanoma diagnosis, BRAF status of primary melanoma, and receiving adjuvant therapy on post metastatic survival. METHODS: We studied melanoma patients presenting to Perlmutter Cancer Center at New York University (NYU) and prospectively enrolled in NYU’s melanoma biospecimen database and followed up on protocol-driven schedule. Patients were stratified by stage at initial melanoma diagnosis as per AJCC 7(th) edition guidelines. Post-metastatic survival was determined using the Kaplan-Meier method, and Cox proportional hazards models were used to assess hazard ratios RESULTS: Three hundred and four out of 3204 patients developed metastatic disease over the time of follow up (median follow up 2.2 years, range 0.08–35.2 years). Patients diagnosed with stage I (n=96) melanoma had longer pmOS (29.5 months) than those diagnosed with stage II (n=99, pmOS 14.9 months) or stage III (n=109, pmOS 15.1 months) melanoma (p=0.036). Initial stage of diagnosis remained significant in multivariate analysis when controlling for LDH and site of metastases (primary diagnosis stage II (HR 1.44, p=0.046), stage III (HR 1.5, p=0.019)). Adjuvant treatment was associated with better survival but BRAF mutation status did not show an association. CONCLUSION: Our data challenge the general assumption that primary melanomas converge upon diagnosis of metastatic disease and behave uniformly. Primary stage of melanoma at the time of diagnosis may be prognostic of outcome, similar to LDH and metastatic disease sites.

Multiple primary melanoma (MPM) has been associated with a higher 10-year mortality risk compared to patients with single primary melanoma (SPM). Given that 3–8% of patients with SPM develop additional primary melanomas, new markers predictive of MPM risk are needed. Based on the evidence that the immune system may regulate melanoma progression, we explored whether germline genetic variants controlling the expression of 41 immunomodulatory genes modulate the risk of MPM compared to patients with SPM or healthy controls. By genotyping these 41 variants in 977 melanoma patients, we found that rs2071304, linked to the expression of SPI1, was strongly associated with MPM risk reduction (OR = 0.60; 95% CI = 0.45–0.81; p = 0.0007) when compared to patients with SPM. Furthermore, we showed that rs6695772, a variant affecting expression of BATF3, is also associated with MPM-specific survival (HR = 3.42; 95% CI = 1.57–7.42; p = 0.0019). These findings provide evidence that the genetic variation in immunomodulatory pathways may contribute to the development of secondary primary melanomas and also associates with MPM survival. The study suggests that inherited host immunity may play an important role in MPM development.

9510 Background: There are no validated blood-based biomarkers to monitor efficacy in pts with advanced melanoma. Lactate dehydrogenase (LDH) is an established prognostic factor; however, it is not normally used to inform treatment decisions. ctDNA at baseline (BL) is associated with a poor prognosis in pts treated with BRAF inhibitors, but no studies have examined the association between serial changes in ctDNA and survival after BRAF and/or MEK inhibitor therapy. Methods: We measured BRAF V600E/K ctDNA at BL and wk 4 in plasma samples from a pooled population of pts with unresectable or metastatic melanoma treated with D or D+T in the phase 3 COMBI-d trial (NCT01584648). We used mutation-specific droplet digital PCR assays; ctDNA results were categorized as positive/negative (pos/neg) using an analytically validated detection threshold of 0.25 copies/mL. Progression-free (PFS) and overall survival (OS) were analyzed in all pts and by BL LDH level (> or < upper limit of normal). Results: BL ctDNA was detectable in 320/345 pts (92.7%) and was not associated with survival. Nearly all pts had a considerable drop in ctDNA after 4 wks of therapy; 201 pts had paired samples (BL and wk 4) and detectable ctDNA at BL. In 80/201 pts (40%) whose ctDNA changed from pos at BL to neg at wk 4, PFS and OS were prolonged vs in 121/201 (60%) whose ctDNA remained pos (median PFS, 12.9 [95% CI, 9.2-20.2] mo vs 7.1 [5.5-8.9] mo; HR, 0.55 [0.39-0.76]; P = .0003; median OS, 28.2 [20.5-48.8] mo vs 14.6 [11.8-18.7] mo; HR, 0.56 [0.40-0.79]; P = .0007). Undetectable ctDNA at wk 4 was associated with prolonged PFS and OS, especially in pts with high BL LDH (Table). Conclusions: Particularly in pts with high LDH, on-treatment ctDNA monitoring may be helpful for early identification of pts likely to benefit from D or D+T. All ctDNA Samples at Wk 4. Clinical trial information: NCT01584648. [Table: see text]

Melanoma is one of the ten most common cancers in the US. Early detection is crucial for survival, but often the cancer is diagnosed in the fatal stage. Deep learning has the potential to improve cancer detection rates, but its applicability to melanoma detection is compromised by the limitations of the available skin lesion databases, which are small, heavily imbalanced, and contain images with occlusions. We build deep-learning-based tools for data purification and augmentation to counter-act these limitations. The developed tools can be utilized in a deep learning system for lesion classification and we show how to build such a system. The system heavily relies on the processing unit for removing image occlusions and the data generation unit, based on generative adversarial networks, for populating scarce lesion classes, or equivalently creating virtual patients with pre-defined types of lesions. We empirically verify our approach and show that incorporating these two units into melanoma detection system results in the superior performance over common baselines., Comment: Accepted to CVPR ISIC Workshop - 2019

BackgroundMelanoma lacks validated blood-based biomarkers for monitoring and predicting treatment efficacy. Cell-free circulating tumour DNA (ctDNA) is a promising biomarker; however, various detection methods have been used, and, to date, no large studies have examined the association between serial changes in ctDNA and survival after BRAF, MEK, or BRAF plus MEK inhibitor therapy. We aimed to evaluate whether baseline ctDNA concentrations and kinetics could predict survival outcomes.MethodsIn this clinical validation study, we used analytically validated droplet digital PCR assays to measure BRAFV600-mutant ctDNA in pretreatment and on-treatment plasma samples from patients aged 18 years or older enrolled in two clinical trials. COMBI-d (NCT01584648) was a double-blind, randomised phase 3 study of dabrafenib plus trametinib versus dabrafenib plus placebo in previously untreated patients with BRAFV600 mutation-positive unresectable or metastatic melanoma. Patients had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. COMBI-MB (NCT02039947) was an open-label, non-randomised, phase 2 study evaluating dabrafenib plus trametinib in patients with BRAFV600 mutation-positive metastatic melanoma and brain metastases. Patients in cohort A of COMBI-MB had asymptomatic brain metastases, no previous local brain-directed therapy, and an ECOG performance status of 0 or 1. Biomarker analysis was a prespecified exploratory endpoint in both trials and performed in the intention-to-treat populations in COMBI-d and COMBI-MB. We investigated the association between mutant copy number (baseline or week 4 or zero conversion status) and efficacy endpoints (progression-free survival, overall survival, and best overall response). We used Cox models, Kaplan-Meier plots, and log-rank tests to explore the association of pretreatment ctDNA concentrations with progression-free survival and overall survival. The effect of additional prognostic variables such as lactate dehydrogenase was also investigated in addition to the mutant copy number.FindingsIn COMBI-d, pretreatment plasma samples were available from 345 (82%) of 423 patients and on-treatment (week 4) plasma samples were available from 224 (53%) of 423 patients. In cohort A of COMBI-MB, pretreatment and on-treatment samples were available from 38 (50%) of 76 patients with intracranial and extracranial metastatic melanoma. ctDNA was detected in pretreatment samples from 320 (93%) of 345 patients (COMBI-d) and 34 (89%) of 38 patients (COMBI-MB). When assessed as a continuous variable, elevated baseline BRAFV600 mutation-positive ctDNA concentration was associated with worse overall survival outcome (hazard ratio [HR] 1·13 [95% CI 1·09-1·18], p

Importance Dermoscopy education in US dermatology residency programs varies widely, and there is currently no existing expert consensus identifying what is most important for resident physicians to know. Objectives To identify consensus-based learning constructs representing an appropriate foundational proficiency in dermoscopic image interpretation for dermatology resident physicians, including dermoscopic diagnoses, associated features, and representative teaching images. Defining these foundational proficiency learning constructs will facilitate further skill development in dermoscopic image interpretation to help residents achieve clinical proficiency. Design, Setting, and Participants A 2-phase modified Delphi surveying technique was used to identify resident learning constructs in 3 sequential sets of surveys—diagnoses, features, and images. Expert panelists were recruited through an email distributed to the 32 members of the Pigmented Lesion Subcommittee of the Melanoma Prevention Working Group. Twenty-six (81%) opted to participate. Surveys were distributed using RedCAP software. Main Outcomes and Measures Consensus on diagnoses, associated dermoscopic features, and representative teaching images reflective of a foundational proficiency in dermoscopic image interpretation for US dermatology resident physicians. Results Twenty-six pigmented lesion and dermoscopy specialists completed 8 rounds of surveys, with 100% (26/26) response rate in all rounds. A final list of 32 diagnoses and 116 associated dermoscopic features was generated. Three hundred seventy-eight representative teaching images reached consensus with panelists. Conclusions and Relevance Consensus achieved in this modified Delphi process identified common dermoscopic diagnoses, associated features, and representative teaching images reflective of a foundational proficiency in dermoscopic image interpretation for dermatology residency training. This list of validated objectives provides a consensus-based foundation of key learning points in dermoscopy to help resident physicians achieve clinical proficiency in dermoscopic image interpretation.

It is well established that survival outcomes for melanoma are highest when these tumors are diagnosed and surgically removed at their earliest stages, prior to the escape of tumor cells to regional and distant anatomic sites. Screening efforts in other malignancies such as colorectal and cervical cancers have yielded benefits in reducing the incidence of invasive cancers and their respective mortalities. Therefore, early detection through broad-based skin cancer screening appears to be a logical step to reduce melanoma mortality. To date, this strategy has not been endorsed by the United States Preventative Services Task Force (USPSTF) due to a lack of conclusive evidence. For example, the initially promising reductions in melanoma mortality observed during a skin cancer screening program in Schleswig-Holstein, Germany, were not replicated in subsequent years in Schleswig-Holstein itself or throughout Germany when the screening program was extended countrywide. The lack of solid evidence demonstrating the benefits of skin screening on melanoma mortality, coupled with the potential harms of screening, and the phenotypic heterogeneity of melanoma raise important concerns regarding the utility of broad-based skin cancer screening programs. Nevertheless, melanoma’s visibility on the skin compared to internal malignancies, and the development of new diagnostic technologies, may help balance the benefits and harms of melanoma screening when considering the varied patient populations in which screening could be deployed. In this presentation we will review current data in the context of developing early-detection strategies to reduce melanoma mortality and/or the cost of care while minimizing potential harms. Citation Format: David Polsky. Opportunities and challenges in melanoma early detection [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr IA13.

Introduction: Serial monitoring of sensitive and specific blood-based biomarkers of disease burden in cancer patients could enable clinicians to adjust treatments at the earliest signs of disease progression when the likelihood of improved outcomes is probably greatest. Such assays require preanalytical, analytical, and clinical validation prior to use. We conducted analytical validation studies of 7 droplet digital PCR (ddPCR) assays to determine their performance characteristics in detecting mutant TERT, BRAF, and NRAS ctDNA. Methods: Probe-based ddPCR assays (Bio-Rad) detecting BRAF V600E, K, NRAS Q61K, R, L, and TERT C228T, C250T were studied. Preanalytical validation experiments using plasma collected in EDTA tubes investigated processing variables including plasma volumes, centrifugation speeds, and DNA extraction efficiencies, and evaluated the necessity of internal controls using spike-in experiments. Analytical validation studies assessed accuracy using plasma from healthy donors spiked with known amounts of mutant nucleosomal DNAs from control cell lines. Potentially interfering substances were evaluated using the AcroMetrix inhibition panel (Thermo Scientific). Precision/reproducibility was assessed using combinations of multiple reagent lots and operators. Assay specificity was determined using templates with different mutations to check cross-reactivity. Limits of Blank (LoB) and Limits of Detection (LoD) were calculated for each assay using 8 replicate wells of healthy donor plasma or mutant cell line DNA, respectively. Three-phase inter-laboratory harmonization was conducted using: 1) extracted DNA, 2) plasmas spiked with control DNAs, and 3) melanoma patient samples shipped in PAXgene Blood ccfDNA tubes (Qiagen). Results: Preanalytical validation yielded a double centrifugation protocol to reduce contaminating large DNA. Assays were highly accurate and precise with 80-90% recovery of spiked-in mutant templates and coefficients of variation (CVs) Conclusion: This ddPCR-based plasma analysis platform demonstrates low background levels in normal plasma, yielding high sensitivities to detect small amounts of ctDNAs with these mutations. The assays also demonstrate highly reproducible results between laboratories. These assays could be integrated into biomarker-driven clinical trials to evaluate their potential to inform more personalized clinical decision-making. Citation Format: Mahrukh M. Syeda, Broderick Corless, Iman Osman, Cindy Spittle, George Karlin-Neumann, David Polsky. Analytical validation of 7 droplet digital PCR assays detecting TERT, BRAF, and NRAS hotspot mutations in plasma-derived circulating tumor DNA (ctDNA) [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A66.

Introduction: Liquid biopsies, especially plasma cell-free circulating tumor DNA (ctDNA), provide a potential opportunity to be a noninvasive biomarker for the diagnosis and monitoring of glioblastoma (GBM) patients. Previously, we detected TERT promoter hotspot mutations (C228T and C250T) in ctDNA of IDH wild-type (IDHwt) TERT promoter mutant GBM patients with 100% specificity using mutation-specific droplet digital PCR (ddPCR) assays. Here, we examine the association between mutant TERT ctDNA levels and clinical outcomes in newly diagnosed GBM patients undergoing chemoradiation. Methods: We analyzed 76 serially collected plasma samples from 17 patients with suspected IDHwt GBM based on MRI before surgery. Twenty mL of whole blood was collected in EDTA tubes at predetermined times: pre- and postoperatively, at the end of chemoradiation, and 1, 3, and 6 months from the end of chemoradiation. TERT promoter mutations C228T or C250T were identified in FFPE tumor samples using ddPCR assays specific for these mutations. Plasma samples were analyzed for the patient’s tumor TERT mutation using the ddPCR assays. The analytically validated thresholds for positive ctDNA detection were 1.5 and 1.7 copies/mL for C228T and C250T, respectively. Results: Sixteen of 17 (94%) IDHwt tumors had TERT mutations (10 C228T, 6 C250T) with MGMT methylated, unmethylated, or unknown status in 10, 5, and 1, respectively. Fourteen of the 16 patients (87.5%) had detectable mutant ctDNA at one or more time points (range 1.66 to 22.13 copies/mL). Of the 2 patients with undetectable ctDNA, one had diffuse and non-avidly enhancing disease and the other only had pre/postop plasma samples collected. Six patients had detectable ctDNA preop, and most had a dominant rim-enhancing mass with additional nonenhancing or enhancing lesion(s). Ten patients had detectable ctDNA up to 4 days postop, half of whom had undergone gross total resection. For 3 of 5 patients for whom there was a question of pseudoprogression versus true progression, ctDNA kinetics matched the clinical outcome. One patient with MGMT unmethylated multifocal GBM achieved ctDNA zeroconversion at 6 months post radiation (RT), and did not progress for another five months. Another patient was negative at all time points until their 3-month post RT follow-up, at which time they developed a recurrence. Another patient achieved zeroconversion at the end of RT but developed a borderline positive ctDNA at 6 months after RT, 2 months before documented radiographic progression. Conclusions: In this pilot, prospective ctDNA monitoring study of IDHwt GBM, TERT mutant ctDNA was detected at one or more time points in the majority of patients. ctDNA kinetics were associated with clinical outcomes for some patients. These data suggest that additional, larger studies could refine how ctDNA monitoring may be used to enhance the clinical management of IDHwt GBM patients. Citation Format: Christine Cordova, Mahrukh M. Syeda, Broderick Corless, Jennifer M. Wiggins, Amie Patel, Sylvia C. Kurz, Malcolm Delara, Zacharia Sawaged, Minerva Utate, Dimitris Placantonakis, John Golfinos, Jessica Schafrick, Joshua S. Silverman, Rajan Jain, Matija Snuderl, David Zagzag, George Karlin-Neumann, David Polsky, Andrew S. Chi. Longitudinal detection of TERT-mutant plasma cell-free circulating tumor DNA in newly diagnosed glioblastoma patients [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A65.

Background Two primary histologic subtypes, superficial spreading melanoma (SSM) and nodular melanoma (NM), comprise the majority of all cutaneous melanomas. NM is associated with worse outcomes, which have been attributed to increased thickness at presentation, and it is widely expected that NM and SSM would exhibit similar behavior once metastasized. Herein, we tested the hypothesis that primary histologic subtype is an independent predictor of survival and may impact response to treatment in the metastatic setting. Methods We examined the most recent Surveillance, Epidemiology, and End Results (SEER) cohort (n = 118 508) and the New York University (NYU) cohort (n = 1621) with available protocol-driven follow-up. Outcomes specified by primary histology were studied in both the primary and metastatic settings with respect to BRAF-targeted therapy and immunotherapy. We characterized known driver mutations and examined a 140-gene panel in a subset of NM and SSM cases using next-generation sequencing. All statistical tests were two-sided. Results NM was an independent risk factor for death in both the SEER (hazard ratio [HR] = 1.55, 95% confidence interval [CI] = 1.41 to 1.70, P < .001) and NYU (HR = 1.47, 95% CI = 1.05, 2.07, P = .03) cohorts, controlling for thickness, ulceration, stage, and other variables. In the metastatic setting, NM remained an independent risk factor for death upon treatment with BRAF-targeted therapy (HR = 3.33, 95% CI = 1.06 to 10.47, P = .04) but showed no statistically significant difference with immune checkpoint inhibition. NM was associated with a higher rate of NRAS mutation (P < .001), and high-throughput sequencing revealed NM-specific genomic alterations in NOTCH4, ANK3, and ZNF560, which were independently validated. Conclusions Our data reveal distinct clinical and biological differences between NM and SSM that support revisiting the prognostic and predictive impact of primary histology subtype in the management of cutaneous melanoma.

Melanoma lacks a clinically useful blood-based biomarker of disease activity to help guide patient management. To determine whether measurements of circulating, cell-free, tumor-associated BRAF(mutant) and NRAS(mutant) DNA (ctDNA) have a higher sensitivity than LDH to detect metastatic disease prior to treatment initiation and upon disease progression we studied patients with unresectable stage IIIC/IV metastatic melanoma receiving treatment with BRAF inhibitor therapy or immune checkpoint blockade and at least 3 plasma samples obtained during their treatment course. Levels of BRAF(mutant) and NRAS(mutant) ctDNA were determined using droplet digital PCR (ddPCR) assays. Among patients with samples available prior to treatment initiation ctDNA and LDH levels were elevated in 12/15 (80%) and 6/20 (30%) (p = 0.006) patients respectively. In patients with RECIST scores